Use of dha, epa or dha-derived epa for treating a pathology associated with cellular oxidative damage

ABSTRACT

The present invention relates to the use of an acid enriched in docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA) or DHA-derived EPA for manufacturing a drug for the treatment of processes that involve associated oxidative damage. In particular, it is for the treatment of processes associated with neurodegenerative, ocular, ischaemic and inflammatory pathology, atherosclerosis, with oxidative damage to DNA and with physical exercise.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 12/158,205 filed Jun. 19, 2008, which is a 371 application ofPCT/EP2006/070016, filed Dec. 20, 2006, which claims priority to SpainApplication Nos. P-200503202 filed Dec. 21, 2005; P-200602417 filed Sep.25, 2006; P-200602418 filed Sep. 25, 2006; and P-200603231 filed Dec.20, 2006, each of which is hereby incorporated by reference in itsentirety.

TECHNICAL FIELD

The present invention relates to the use of an acid enriched indocosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA) or DHA-derivedEPA for manufacturing a drug for the treatment of processes that involveassociated oxidative damage.

BACKGROUND

The omega-3 fatty acids are necessary for maintaining cellularfunctional integrity, and are necessary in general for human health.Docosahexaenoic acid (22:6 n-3, DHA), an important omega-3 component offish oil and of marine algae, is concentrated in the brain, in thephotoreceptors and in the synapses of the retina. DHA-enriched diets areinitially metabolised by the liver and afterwards distributed via thelipoproteins in the blood in order to meet the needs of the variousorgans. The administration of DHA leads to an increase of itsconcentration at tissue level, inducing also an increase in theconcentration of omega-3 eicosapentaenoic acid (EPA) which in linkedmetabolically, whereas the administration of EPA only increases itsconcentration decreasing that of DHA at cell level.

In general, the DHA is incorporated into the phospholipids of the cellmembrane, which have effects on its composition and functionality, onthe production of reactive oxygen species (ROS), on membrane lipidoxidation, on transcription regulation, on the biosynthesis ofeicosanoids and on intracellular signal transduction. Furthermore, inthe central nervous system, the DHA is involved in the development ofthe learning capacity related to the memory, in the excitable functionsof the membrane, in biogenesis of the photoreceptor cells and intransducing the signal dependent upon quinase protein. A potentialdietary therapy would be based on correcting the optimum levels ofomega-3 fatty acids to prevent certain pathologies from originating orprogressing, such as inflammatory pathologies, tumoral processes,cardiovascular diseases, depression and neurological disorders.

In the central nervous system, both the brain and the retina show anunusual capacity for retaining DHA, even under situations of veryprolonged dietary deficiencies of omega-3 fatty acids. Several studieshave described the protective effect of DHA on neurones, in which it ispresent in very high levels. For example, it is involved in protectingthe neuronal cells from death by apoptosis. Recently, it has been shownthat DHA, found in reduced amounts in the hippocampus of rats ofadvanced age, is capable of protecting primary cultures of said cellsagainst the cytotoxicity induced by glutamate.

In the photoreceptors of the retina, DHA has also been shown to modulatethe levels of the pro- and anti-apoptotic proteins of the Bcl-2 family.The external segments of the retinal photoreceptor contain rodopsin, aswell as a higher DHA content than any other type of cell. The DHA isconcentrated in the phospholipids of the photoreceptor segment disc'souter membranes. Retinal dysfunctions have been observed underconditions of reduction of optimal DHA concentration. The retinapigmentary epithelial cell (RPE) plays a very active role in DHAtake-up, conservation and transport. The high DHA content in thephotoreceptor and in the RPE cells is mainly linked to domains in themembrane with physical characteristics that contribute to the modulationof receptors, ionic channels, carriers, etc., while it also appears toregulate the concentration of phosphatidilserine.

It is unknown to date if these effects are entirely mediated by the DHAitself or by any metabolic derivatives. Certain derivatives of DHA havebeen identified in the retina. Although the enzymes involved in thesynthesis of said derivatives have not been identified precisely, somerecent results suggest the participation of an A₂ phospholipase (PLA₂)followed by a lipoxygenase (LOX). The PLA₂ releases the DHA from themembrane phospholipids and the LOX converts it into its metabolicallyactive derivatives.

The reactive oxygen species (ROS) are produced during normal cellularfunctioning. The ROS include the superoxide anion, hydrogen peroxide andthe oxydril radical. Their high chemical reactivity leads to theoxidation of proteins, of DNA or of lipids. The superoxide dismutase(SOD), the catalase (CAT) and the glutation peroxidase (GPx) are theprimary antioxidant enzymes that protect against the molecular andcellular damage caused by the presence of ROS. The oxidative stressactivates many metabolic channels; some are cytoprotective, while otherslead to death of the cell. Recent studies indicate that an imbalancebetween ROS production and breakdown is a significant risk factor in thepathogenesis of many illnesses, in some cases related to a deteriorationof the antioxidant system.

The DHA is presented as a target of the ROS that produces damage to thecell of the photoreceptor and to the RPE. The retinal degenerationinduced by light promotes loss of DHA in the photoreceptors. Forexample, when the RPE cells are damaged or die, photoreceptor functiondeteriorates because the RPE cells are essential for its survival. Thus,death of the RPE cell under the effect of oxidative stress leads to adeterioration of eyesight, particularly when the cells of the macula areaffected, since it is responsible for eyesight acuity. Thepathophysiology of many retinal degenerations (e.g., maculardegenerations related to age and to Stargardt disease) involvesoxidative stress that leads to RPE cell apoptosis. Indeed, RPE cellapoptosis appears to be the dominant factor in the macular degenerationobserved with age. Such studies suggest that said cells have developedhighly effective antioxidant mechanisms to protect themselves from theirhigh DHA content and show notable adaptive capacity.

Furthermore, the relationship between the free radicals and ageing isperfectly well accepted, based on the evidence that free radicalsproduced during aerobic respiration cause oxidative damage thataccumulates and leads to a gradual loss of the homeostatic mechanisms,interference in gene expression patterns and a loss of the cell'sfunctional capacity, leading to ageing and death. An interrelationexists between the generation of oxidants, antioxidant protection andrepair of the oxidative damage. Many studies have been carried out todetermine whether antioxidant defences decline with age. These haveincluded analysis of the main components thereof: activity or expressionof the SOD, CAT, GPx enzymes, glutation reductase,glutation-S-transferase and the concentration of compounds of lowmolecular weight with antioxidant properties. For example, anover-expression of SOD and CAT in Drosophila melanogaster increases lifeexpectancy by 30% and reduces damage by protein oxidation. In thiscontext, in vitro and in vivo exposure of cutaneous tissue to UV raysgenerates free radicals and other reactive oxygen species, leading tocellular oxidative stress, documented as contributing significantly toageing. Excessive exposure of the skin to ultraviolet radiation can giverise to acute or chronic damage. Under acute conditions erythema orburns can be produced, while chronic over-exposure increases the risk ofskin cancer and ageing. Moreover, it is known that the cutaneous cellscan respond to acute or chronic oxidative stress by increasingexpression of a variety of proteins, such as the enzymes involved inmaintaining cell integrity and resistance to oxidative damage.

In the art, it is well known that telomeres are non-coding DNA regionslocated at the ends of eukaryotic chromosomes. These are constituted byhighly conserved DNA sequences, repeated in tandem (TTAGG)_(n), andassociated proteins, and have a special structure which hinders theligation to the ends of other chromosomes, preventing the telomericfusion. They have an essential role in the preservation of thechromosomic integrity, protecting the coding DNA from the enzymaticaction and its degradation, contributing to the maintenance of thechromosomic stability.

In contrast with coding sequences which have a semiconservativereplication, the telomeres undergo a progressive loss of its repetitivesequences during the successive cell division. Nowadays, it isconsidered that a minimum telomeric length is required in order to keepthe telomere function and when these reach a critical size they havedifficulties for the division in the mitosis, generating telomericassociation (TAS) and chromosomic instability. Said chromosomicinstability would be associated with an increase in the probability ofproducing errors capable of generating significant genetic changes.

Owing to the multiplicity of double bonds, the omega-3 fatty acids areconsidered to be molecular targets for generation and propagation offree radicals during the oxidative stress processes related togeneration of lipidic peroxides. Contradictory results have beenobtained, however, in various studies of susceptibility to oxidativestress owing to dietary supplements of omega-3 fatty acids. Some studiesin humans have shown increased oxidation of the LDL, while others havefound no such effect. In studies with animals, treatment with omega-3fatty acids has been found to lead to increased or reducedsusceptibility to oxidation of the LDL. On the other hand, anover-expression of the genes involved in the antioxidant defence systemhas been found in the livers of mice fed on a fish-oil-enriched diet forthree months.

Furthermore, various in vitro studies with a cellular line of glyalorigin have shown that membranes rich in omega-3 fatty acids are moresusceptible to oxidative damage. Long-term supplementation of thesecells with high concentrations of DHA resulted in increased levels oflipidic peroxides in the culture medium, and a higher percentage of celldeath due to apoptosis induced by exposure to hydrogen peroxide. It hasalso been shown, however, that intra-amniotic administration of ethyldocosahexaenoate reduces lipidic peroxidation in the foetal brains ofrats. It has been suggested that this response is due to a free-radicalsequestering effect via activation of antioxidant enzymes. An increasein the antioxidant capacity of the brain is important for the primaryendogenous defence against oxidative stress, because the brain isrelatively rich in polyunsaturated fatty acids and relatively poor inantioxidant enzymes.

These contradictory results suggest that the hypothesis based on thepremise that oxidation of a fatty acid increases with the number ofdouble bonds has no in vivo applicability, since other potentialmechanisms may act to reduce oxidative damage, such as athree-dimensional structure of the omega-3 fatty acids in the lipids andlipoproteins of the membrane that make the double bonds less susceptibleto an attack by the ROS, an inhibition of pro-oxidant enzymes such asPLA₂ or a greater expression of antioxidant enzymes.

On the other hand, the idea of associating physical exercise with theproduction of free radicals comes from early 80s due to the observationof the damage in membrane lipids during ischemia-reperfusion events inhypoxic tissue (see Lovlin et al., Eur. J. Appl. Physiol. Occup.Physiol. 1987, 56 (3) 313-6). At the same time, an increase in theGSSH/GSH ratio was observed in rat muscle cells (see Lew H. Et al. FEBSLett, 1985; 185(2): 262-6, Sen C K et al., J. Appl. Physiol. 1994;77(5): 2177-87) as well as in human blood (see MacPhail Db et al., FreeRadic Res Commun 1993; 18(3): 177-81, Gohil K. et al. J. Appl. Physiol.1988 January; 64(1): 115-9). Free radicals also affect DNA and acutephysical exercise increases damage in DNA, as evidenced by the increaseof 8-OxodG. Exhausting physical effort (running a marathon) causesdamage in DNA which is evident for some days after the trial and alsocauses damage in immunocompetent cells (which can be associated with theimmune decrease shown in sportsmen after such a trial).

However, other authors did not observe any effects (except for minordamage) after swimming for 90 minutes, running for 60 minutes or makingan exhausting effort by rowing. At the same time, researches on trainedand non-trained sportsmen did not find any difference in the urinaryexcretion of 8-oxo-dG, even those finding such damage, considered to besecondary to subsequent reactions to the effort and not to the action ofexercise over the DNA in acute way.

The event of intensive physical exercise producing oxidative stress isvery well known in the art, but its origin is not well determined yet.

Studies carried out with n-3 fatty acids related to sports performancewere focused on the antiinflammatory effect and, indeed, first assaystried to find the possible action of these nutrients improving thealveolar-capillary absorption by diminishing the intensive physicalexercise-induced broncoconstriction. In that regard, Mickleboroughproved that after administering 3.2 g EPA and 2.2 g DHA regimeproinflammatory cytokines were attenuated by diminishing the presence ofTNF-α and IL-1β in an elite athlete, along with a decrease in thebroncoconstriction. Walser related n-3 fatty acids vascular effects topositive effects in people showing intolerance to physical exercise. Inthat regard, van Houten et al. studied that a n-3 fatty acid highingestion was associated with a better recovery in patients carrying outa cardiac rehabilitation after a coronary syndrome.

The absence of positive results in the physical performance in theanalyzed studies is due to the evaluation of patients, not healthypeople, and what it has been searched are vascular and inflammatoryeffects.

At the same time, researches have been carried out based on thefollowing theoretical concept: increasing free fatty acids in plasmaabove 1 mmol/L (occurring when glycogen is used up), the competence withtryptophan transport makes this to be increased with the subsequentserotonine increase, a neurotransmitter related to the so-called“central fatigue” in long duration sports. In that regard, it is knownthat n-3 fatty acids diminish the amount of free fatty acids in plasmaprobably by up-regulating the fatty acid oxidation by activating thetranscription nuclear factor PPARα. However, these assays were notsuccessful, since Huffman (2004) by using a dose regime of 4 g of n-3fatty acid (500 g capsules containing 300 mg EPA and 200 mg DHA) carriedout a study in both sex runners without finding any decrease in free TRPnor a less perception of effort, nor any statistically performanceincrease in the performance, although there was a statistical tendencyfor improving the performance in subjects whom n-3 fatty acid wereadministered, leaving open the possibility to authors that the cause ofdiminishing the statistical power for the study was the low number ofsubjects studied (5 men and 5 women).

Another subsequent research wherein the efficacy of n-3 acids related tothe performance was evaluated did not find any significant differencesusing maize oil as a placebo. Raastad administering 1.60 g EPA and 1.04g DHA per day for several weeks, did not find any improvement infootball players (see, Raastad et al. Scand J. Med Sci Sports 1997;7(1): 25-31).

On the other hand, it is known that free fatty acids interfere with theuse of glucose in the muscle, since its analogues at intracellularlevel, acyl-CoA, in the mitochondria inhibit the pyruvate dehydrogenase(inhibition by product), furthermore, stimulates glycogenolysis andglyconeogenesis, causing a smooth hyperglycemia during fasting, indeed,the continuous administration of polyunsaturated fatty acids duringfasting helps to maintain glycemia, by maybe activatingglucose-6-phosphatase at a hepatic level. It is also known that acomposition of fatty acids in the muscle alters insulin sensitivity,showing that a high content of polyunsaturated fatty acids in plasmaticmembrane improves insulin sensitivity and a high content of saturatedfatty acids produces the opposite effect.

Exercise increases glucose uptake, capillary perfusion, glycogensynthesis rate and insulin sensitivity. During muscular contractionchanges are produced in temperature, intracellular pH, ATP/ADP ratio, aswell as Ca⁺⁺ intracellular concentration and other metabolites whichcould act as messengers in the cellular functioning regulation withexercise. In this regard, Ca⁺⁺ regulates a great amount of intracellularproteins, including calmodulin kinase, protein kinase C (PKC) andcalcineurin which are important intermediates in the signals ofintracellular transduction. During aerobic exercise, acetyl-CoAcarboxylase is deactivated by AMP kinase (AMPK) which leads to a drop inmalonyl-Coa levels, deinhibiting carnitine palmitol transferase with theresulting increase of fatty acid transport within the mitochondria (thuspromoting fatty acid oxidation).

AMPK activation effects probably include stimulation of GLUT4 andhexokinase expression, as well as mitochondria enzymes. However,surprisingly, AMPK activation is not the unique way (independent ofinsulin) wherein the exercise increases the response to glucose inskeletal muscle. See Mora and Pessin, J. Biol. Chem. 2000; 275 (21):16323-16328, showed that an increase in the glucose response in themuscle, indeed, there are several transcription factors such as MEF2Aand MEF2D activating GLUT4 and those factors are activated by exercise.

An increase in intramuscular lipids is common in obesity states andphysical training, but the result is that for obese people is associatedwith insulin resistance, whereas in sportsmen the great activity ofcarnitine palmitol transferase makes fatty acids undergo beta oxidation.There are strong evidences that a rich diet in n-3 fatty acid, even withan increase of glycemia and insulinaemia (signals of insulinresistance), act at a insulin receptor level maintaining the level ofGLUT-4 protein translocation, which has specifically showed with DHA(see, Jaescchke H. Proc. Soc Exp Biol. Med 1995; 209: 104-11).

DESCRIPTION OF THE INVENTION

The present invention concerns the unexpected discovery that theadministration of docosahexaenoic acid (herein also referred to as DHA)or eicosapentaenoic acid (EPA) or DHA-derived EPA, whether in free formor incorporated into a triglyceride, among others, acts as a cellularantioxidant.

In this way and taking into account the metabolic relation between DHAand EPA (retroconversion of DHA to EPA), all effects disclosed observedpreviously for the administration of DHA must be applicable to mixedsystems DHA/EPA or even to monocomponent systems of EPA, even though EPAis not named specifically.

An object of the present invention is therefore the use ofdocosahexaenoic acid for the manufacturing of a pharmaceuticalcomposition for the treatment of cellular oxidative damage.

Another object of the present invention is the use of docosahexaenoicacid (DHA) at a specific position of the glycerol backbone, the tworemaining positions of the glyceride being also specified in theircomposition for the treatment of cellular oxidative damage.

A further object of the present invention is the use of docosahexaenoicacid (DHA) for manufacturing a composition for the treatment of thecellular oxidative damage at DNA level. In particular, the use ofdocosahexaenoic acid has the application as a protective agent in thenatural process of telomere shortening and as an inhibitory agent ofpremature senescence in a treatment of cellular oxidative damage.

It is also an object of the present invention the use of docosahexaenoicacid for manufacturing a composition for the treatment of cellularageing and hereditary pathologies associated with disorders in themitochondrial respiratory chain, as well as a composition for treatingDown's Syndrome.

A further object of the present invention is the use of docosahexaenoicacid (DHA) for manufacturing a composition for the treatment of thecellular oxidative damage associated with physical exercise. Inparticular, the use of docosahexaenoic acid has application as anenhancer agent in the sports performance and as a regulating agent ofblood glucose levels during physical effort.

It is also an object of the present invention the use of docosahexaenoicacid for manufacturing a composition for enhancing sports performance,as well as a composition for maintaining blood glucose levels afterphysical exercise by means of, mainly, the administration of a food, adairy product or any suitable administration form typically used bypeople when doing physical exercise.

In the present invention, the expression “cellular oxidative damage”means any process that involves an imbalance between the generation anddegradation of cellular oxidant species of endogenous or exogenousorigin.

Surprisingly, the inventors of the present invention have found that DHAis capable of inhibiting the production of reactive oxygen species(ROS), whether related to a dependent induction of peroxides orsuperoxides. More specifically, it reduces the production of superoxideanion and therewith of all the derived species produced in the oxidativecascade, such as for example a highly significant reduction of lipidicperoxidation. Furthermore, an increase in antioxidant enzyme activitywas found, which suggests an adaptation of the cell by inducing theexpression of antioxidant agents, basically enzymes, and by repressingthe expression of pro-oxidant agents such as the A₂ phospholipase.

In one embodiment of the present invention, said docosahexaenoic acid isincorporated into a monoglyceride, diglyceride, triglyceride,phospholipid, ethyl ester or free fatty acid. Preferably, saiddocosahexaenoic acid is incorporated into a triglyceride.

In the present invention, “docosahexaenoic acid incorporated into aglyceride” is taken to mean a monoglyceride, diglyceride, triglyceride,phospholipid, with at least one of the three positions esterified with adocosahexaenoic acid and, optionally, at least one of the remainingesterified positions further with one acid selected from a short-, mid-or long-chain fatty acid and a phosphoric acid. Preferably, saidglycerol is a triglyceride.

The choice of triglyceride as chemical structure of the DHA is based ondata taken from a study which compared the bioavailability of fouromega-3 acid concentrates in the form of ethyl esters, phospholipids,free fatty acids and triglycerides following oral administration, whichdemonstrated that the re-esterified triglycerides presented a higherbioavailability than the other preparations.

In a preferred embodiment of the present invention, said docosahexaenoicacid is found in a percentage by weight of between 20 and 100% inrelation to the total fatty acids, preferably between 40 and 100% inrelation to total fatty acids, and more preferably said docosahexaenoicacid is in a percentage by weight between 66 and 100% in relation tototal fatty acids.

In another preferred embodiment, said docosahexaenoic acid isincorporated into at least one specific position of a glycerol via anester bound, a structured lipid, for manufacturing a pharmaceuticalcomposition for the treatment of cellular oxidative damage.

Such a glycerol may further comprise at least one fatty acid and/or onephosphoric acid so that said docosahexaenoic acid being incorporatedinto a position selected from sn-1, sn-2 and sn-3, may further comprise,optionally, at least one acid selected from a short- and/or mid-chainfatty acid and a phosphoric acid, and when incorporated into the sn-2position may further comprise, optionally, at least one acid selectedfrom a fatty acid and a phosphoric acid.

In this regard, when referring to the term optionally, it should beunderstood that said docosahexaenoic acid incorporated into a positionselected from sn-1, sn-2 and sn-3 may or not further comprise at leastone acid selected from a short- and/or mid-chain fatty acid and aphosphoric acid, or otherwise that said docosahexaenoic acidincorporated into the sn-2 position may or not further comprise at leastan acid selected from a long-chain fatty acid and a phosphoric acid.

Surprisingly, the inventors of the present invention have found that theuse of structured glycerols wherein the position of the docosahexaenoicacid has been selected and the composition of the rest of the compoundbound to the glycerol, leads to an unexpected increase, at least twiceor even thrice, the therapeutic efficiency of the use of docosahexaenoicacid for manufacturing a pharmaceutical composition for the treatment ofcellular oxidative damage.

The common definition relates to fats containing fatty acids located inspecific positions in the glycerol backbone. By similarity with the invivo fatty acid biodistribution, the long-chain polyunsaturated fattyacids (PUFAs) are located preferably in the sn-2 position of theglycerol and taking into account the intestinal absorption process,triglycerides are hydrolized by lipases to free fatty acids, di- andmonoglycerides, from which the free fatty acids and sn-2 monoglyceridesare absorbed directly by intestinal epithelial cells, named enterocytes.

By using docosahexaenoic acid incorporated into a specific position ofthe glycerol backbone, via an ester bound, provides an increasedbioactivity, an increased antioxidant protection at the same molarpercentage in respect with the whole amount of fatty acids present and adiminished dependency on the administration dosage in respect with theantioxidant effect of the docosahexaenoic acid in the glyceride.

Advantageously, the inventors of the present invention have found thatthe use of docosahexaenoic acid incorporated into a position of theglycerol selected from sn-1, sn-2 and sn-3, and optionally said glycerolfurther comprising at least one acid selected from a short- and/ormid-chain fatty acid and a phosphoric acid, provides an increasedbioactivity, an increased antioxidant protection at the same molarpercentage in respect with the whole amount of fatty acids present and adiminished dependency on the administration dosage in respect with theantioxidant effect of the docosahexaenoic acid in the glycerol.

Also advantageously, the inventors of the present invention have foundthat the use of docosahexaenoic acid incorporated into a sn-2 positionof a glycerol and optionally said glycerol further comprising at leastone acid selected from a long-chain fatty acid and a phosphoric acid,provides also an increased bioactivity, an increased antioxidantprotection at the same molar percentage in respect with the whole amountof fatty acids present and a diminished dependency on the administrationdosage in respect with the antioxidant effect of the docosahexaenoicacid in the glycerol.

Preferably, acids also present in a glycerol with the docosahexaenoicacid will be short-chain fatty acids (C1-C8) or mid-chain fatty acids(C9-C14) or a phosphoric acid, since these have no functional activity,but only energetic activity and, therefore, will not compete with thedocosahexaenoic acid.

Therefore, still more preferably, the present inventions relates to theuse of docosahexaenoic acid incorporated into a glycerol wherein one ofthe positions sn-1 and sn-3 are free or occupied by a mid-chain fattyacid (C9-C14) or short-chain fatty acid (C1-C8) or a phosphoric acid andin which sn-2 position is occupied by functional DHA. Thus, a stillhigher increase of DHA is achieved since it is more efficiently absorbedin the intestinal cells.

Therefore, the synthesis of structured glycerides wherein thedocosahexaenoic acid has been incorporated into any position of theglycerol when it does not compete with other fatty acids and wherein theDHA has been incorporated into the sn-2 position of the glyceride whenit competes with at least one fatty acid, shows improvements related toits antioxidant effect and, therefore, it is a preferred way formanufacturing a composition for the treatment of the oxidative cellulardamage.

The inventors of the present invention have found that a cell enrichedwith a composition with DHA, in accordance with the invention, is betterprepared to face up to a new situation of oxidative stress and thus tominimise the adverse effects that can derive therefrom. That is, thepresence of the DHA in the biomembranes induces a cellular adaptiveresponse to the oxidative stress. Adaptive response is a cellularphenomenon by which exposure to a toxic agent (in sub-lethalconcentrations) provokes a cellular response which will subsequentlyprotect the cell against the deleterious effects of that same toxicagent at lethal concentrations, or, put another way, it is a beneficialeffect unleashed by a low level of exposure to an agent that is harmfulat high levels.

Administration of DHA has the following substantial advantages:

a) Increased cellular antioxidant activity;

b) Absence of cellular cytotoxicity at the dosages administered;

c) Absence of significant alterations to cellular oxidant status at thedosages administered;

d) Adaptive cellular antioxidant activity.

Due to all the above, in a preferred embodiment the present inventionrelates to the use of docosahexaenoic acid for manufacturing apharmaceutical composition for treating a pathology associated withcellular oxidative damage, said pathology being a neurodegenerativepathology, preferably selected from the group that comprises: multiplesclerosis, Alzheimer's disease, Parkinson's disease, amiotrophic lateralsclerosis and muscular dystrophy, among others.

In another embodiment of the present invention, the pathology associatedwith the oxidative damage is an ocular pathology, preferably oneselected from the group that comprises pigmentary retinosis, maculardegeneration and cataracts, among others.

In yet another embodiment, the pathology associated with the oxidativedamage is an ischaemic pathology, particularly a myocardial infarct,cerebral infarct, etc.

In yet another embodiment of the present invention, the pathologyassociated with the oxidative damage is an inflammatory process,preferably selected from the group comprising arthritis, vasculitis,glomerulonephritis and eritomatose lupus, among others.

In another preferred embodiment, the pathology associated with theoxidative damage is atherosclerosis.

Another aspect of the present invention is the use of DHA as aprotective agent in the natural process of telomere shortening and as aninhibitory agent of premature senescence.

The mechanisms producing telomeric associations (TAS) are still unknownbut the authors of the present invention suggest that this could beassociated with a deficit in the activity of enzyme telomerase whichsynthesizes the repetitive sequences of DNA characteristic fortelomeres, thereby stabilizing the length thereof.

The telomerase is very active in foetal cells, but has not much activityin adult tissue cells. TAS have seldom found in normal cells, but theyhave been observed in infected cells by virus or tumour cells.

It has been observed that there is a progressive reduction in the numberof in vitro telomeric repetitions, as well as in function of cellularageing, in vivo, which is associated with an inhibition of thetelomerase activity in the senescence. Likewise, the authors of thepresent invention have studied the telomeric length in fibroblasts andlymphocytes from centenary healthy persons, having found a telomericshortening during the in vitro propagation of the fibroblasts, as wellas a reverse correlation between the telomeric lengths and the donor'sage.

Although the telomere shortening occurs naturally with the cellularreplication, a premature senescence and breakages of telomeres wheninducing oxidative damage in the DNA have been observed. The telomeresare more sensitive to oxidative damage and their breakages are lessefficiently repaired than other parts of the genome. This leads to anaccumulation of telomeric damage which produces a faster shorteningduring the DNA replication reducing the cellular replicative lifeexpectancy. The reactive oxygen species (ROS), particularly superoxideanions, hydrogen peroxide and oxidril radicals, can accelerate thelosses in the telomeres during the replication of some cellular types,even though they also induce premature senescence regardless of thetelomere shortening.

Surprisingly, the authors of the present invention have found that theuse of docosahexaenoic acid for the treatment of the cellular oxidativedamage at DNA level allows to reduce the shortening rate of thetelomeres and, therefore, inhibit the cellular senescence.

The present inventors have found a reverse correlation between theshortening rate of telomeres and the cellular antioxidant capacity inmore than 20 fibroblasts human strains. Most of the cellular parametersof these prematurely aged fibroblasts are the same as the normal ageingof these cells (morphology, accumulation of lipofuscin and changes inthe genic expression). The fibroblasts with a lower antioxidant defenceshorten their telomeres faster and vice versa. The shortening rate ofthe telomere is higher in cells with a lower antioxidant defence.Furthermore, free radical scavengers reduce the shortening rate of thetelomere.

These data are in concordance with those showing an important role ofthe antioxidant enzymes, glutation peroxidase and superoxide dismutase,in the shortening rate of telomeres in human fibroblasts. These datesprove that the length of telomeres is determined mainly by the relationbetween the oxidative stress and the cellular antioxidant defencecapacity. Thus, the length of age-dependent telomeres is an accumulativemeasurement of the history of the oxidative damage that a cell hasundergone along its life.

A correlation between oxidative stress and shortening rate of telomereshas been shown for hereditary pathologies associated with disorders inthe mitochondrial respiratory chain and for Down's Syndrome.

Therefore, the existent relation between the oxidative cellular damagein DNA and the telomere shortening and its effect in the cellularsenescence allow to use the docosahexaenoic acid as a powerfulprotective agent in the natural process of telomere shortening and as aninhibitory agent of premature senescence.

On the other hand, the use of enzymes for the production of omega-3fatty acid-enriched oils has several advantages in respect with othermethods based on chemical synthesis and subsequent processes ofpurification (chromatographical separations, molecular distillation,etc.). The latter require extreme conditions of pH and high temperatureswhich could partially destroy all double bounds all-cis of omega-3 PUFAsby oxidation, by cis-trans isomerization or migration of double bound.The soft conditions used in enzymatic synthesis (temperature lower than50° C., pH 6-8 and less chemical reagents) provides a syntheticalternative conserving the original structure of omega-3 PUFAs with anincrease in the structural selectivity in the acylglycerides, consideredto be the most favourable chemical structure from a nutritional point ofview.

The pharmaceutical composition comprising DHA can be found in the formof an oil or an emulsion, which can be administered by oral, sublingual,intravenous, intramuscular, topical, subcutaneous or rectal routes, oreven by merely bringing the active ingredient of the microemulsion ofthe invention in liquid or vapour form into contact with the olfactoryorgans situated at the entrance of the respiratory tracts. Thus, theadministration can be carried out by spraying, misting or atomisation ofthe microemulsions or by inhalation.

Optionally, said pharmaceutical composition further comprises a secondactive ingredient.

Similarly, the pharmaceutical composition comprising DHA can be used inthe food industry for the purpose of enriching food products (e.g.lactic products such as yoghurts, milk, etc) with a natural antioxidantagent such as DHA.

Therefore, in another embodiment of the present invention saidpharmaceutical composition is administered to a patient who is alreadyreceiving a treatment against a pathology associated with oxidativedamage.

Another object of the present invention is the use of DHA as an enhanceragent in the sports performance and as a regulating agent of bloodglucose levels during physical effort.

In this way, the authors of the present invention have surprisinglyfound that the use of said docosahexaenoic acid during physical exerciseleads to an increase in the sports performance maintaining blood glucoselevels (glycemia) after such physical exercise (without administeringcarbohydrates).

On this context of the present invention, by “amateur” or “non-competingsportsmen” is taken to mean any person doing physical exercise in asporadic way and non-professionally. And by “competing sportsmen” or“trained sportsmen” is taken to mean any person doing physical exercisein a regular way and/or at professional level. Likewise, the terms“physical exercise” and “physical effort” are used in an equivalent andexchangeable way, as well as the term “sportsmen” is used for men andwomen.

Sports Performance

In order to evaluate sports performance there are several parameterswhich allow to give a valoration about the improvement of such a sportsperformance.

In sportsmen doing aerobic sports an increase in the performance isconsidered when there is an increase of the percentage of oxygen maximumconsumption % VO_(2max) in the UV 2 (anaerobic threshold), since VO₂maxhardly increases during a competitive season in very well trainedsportsmen. Little changes in the percentage of VO_(2max) in thethreshold are data directly related to an increase in the performance.

The present inventors have shown that a statistically significantincrease of the oxygen consumption (VO₂), both absolute (p<0.019) andrelative (p<0.036) values to the weight in the ventilatory threshold 2when comparing basal triangular effort trials with those carried outafter four months of treatment with DHA. The increase of this parameteris shown for both competing cyclists (p<0.047) and non-competingcyclists, being the difference in the latter non statisticallysignificant (FIG. 24)

Another parameter related to an increase in the sports performance isthe increase in the cardiac frequency wherein the UV2 of the efforttrial is set, since in case the cardiac frequency increases in theanaerobic threshold, the sportsmen are considered to be capable ofslightly increasing its ability of keeping the aerobic metabolism inhigher intensities. The present inventors have observed an increase inthe cardiac frequency in UV2 for p=0.082 when comparing said parameterobtained in the basal trial with that obtained in the triangular trialafter 4 months consuming DHA. These dates are notably shown (p<0.017) inthe subgroup of cyclists with a high competitive level (FIG. 25).

In this regard, there is an increase in the time needed to reach thestatistically significant UV2 (FIG. 26).

Finally, the cardiac frequency for the same effort level is lower ifsportsmen are aerobically trained. The present inventors have seen thatin cyclists being administered with DHA cardiac frequency decreases in astatistically significant way (p<0.043) when comparing these data forboth trials at the point when sportsmen consume 2000 ml O₂/min (FIG. 27)

It can be concluded from these studies that in sportsmen taking DHA for4 months an increase in the consumption of oxygene, absolute andrelative, in the UV2 (p<0.008 and p<0.015, respectively), an increase inthe charge corresponding to the UV2 (p<0.063) and a decrease in thecardiac frequency when sportsmen presents an oxygene consumption of 2000ml/min (p<0.062) have been observed. All these are parameters indicatingan increase in the sports performance after taking 2.1 g DHA/24 h (6capsules of 500 mg at 70% by weight), distributed in 3 daily dose for 4months. Said quantities are expressed by way of example andnon-limitative of the present invention.

Several biochemical variables related to oxidative damage were alsoanalyzed after effort trials.

1.—Plasma Total Antioxidant Capacity (PTAC). There is a general andsignificant statistically increase of PTAA (p<0.05) while carrying outrectangular trials. These increases are higher in sportsmen after beingadministered DHA for three weeks, both considering as a whole or ascompeting cyclists, without showing any difference between basal trialand trial realized after consuming DHA for three weeks by amateursportsmen (FIG. 28)

2.—Malonyldialdehyde (MDA). MDA is the mostly obtained product afterreacting lipidic peroxides produced by oxidative stress withthiobarbituric acid. It is shown that a significant increase ofoxidative damage to plasmatic lipids while carrying out all efforttrials (p<0.035). After DHA ingestion for 3 weeks, oxidative damage tolipids while carrying out the effort trial is lower than that at thebeginning (p<0.05). This difference is much more important for trainedsportsmen than for amateur sportsmen (FIG. 29)

3.—8-oxo-7,8-dihydro-2-′-deoxyguanosine (8-oxodG). 8-oxodG is anoxidative stress biomarker. There is an increase of oxidative damage tothe DNA while carrying out rectangular effort trials (p<0.011). Thisoxidative damage diminishes after administering DHA for 3 weeks(p<0.035). This decrease in the oxidative stress is more important innon-competing cyclists than in competing cyclists, this difference notbeing statistically significant (FIG. 30)

Glycemia Studies

In order to study blood glucose levels a rectangular effort trial wascarried out on a bicycle roller with a maximum charge maintainedequivalent to a rate corresponding to 75% of VO₂max calculated over themaximum triangular effort trial, maintaining the slope constant at avalue of 2%. The time for the trial is 90 minutes and the consumption ofwater through the same is carried out ad libitum.

Since beverage with carbohydrates were not ingested, an hypoglycemia wasexpected. This hypoglycemia of second extraction (twenty minutes afterthe end of the trial in respect to the starting sample obtained twentyminutes before the start), is shown in the first effort trial, as it wasexpected. However, data obtained after the DHA administration for fourmonths show a statistically significant glycemia maintainement, whichwas not observed previously and it represents a surprising finding inthe realized research.

Generally, a statistically significant decrease (p<0.0009) of serumglucose levels throughout rectangular effort trial is observed. However,the behaviour is different depending on the type of sportsmen to beanalyzed (p<0.003): in the case of usually competing cyclists, there wasno significant variation in the decrease of glycemia during the trials,but in case of amateur cyclists, the decrease of glycemia during thebasal trial is higher than in usually competing cyclists and aftertaking DHA for 3 weeks or four months, said decrease virtuallydisappears (FIGS. 31, 32 and 33).

The existence of normoglycemia during the effort trial at 75% of VO₂maxfor 90 minutes without drinking the beverage with carbohydratesrepresents a finding which connects the behaviour of DHA during aphysical effort with that observed and above-mentioned in relation withthe increase of insuline sensitivity. In this regard, Goodyear and Kahn(1998) concluded that the molecular mechanisms underlying the responseto glucose in the skeletal muscle by insuline or exercise, are differentafter the publication in 1997 (Winder and Hardie) about the fact AMPK(AMP—activated protein kinase) was high in fibers Iia during exercise,considering that AMPK has a pleiotropic effect inhibiting acetyl-CoAcarboxylase and promoting glucose transport among other actions.Perhaps, this may explain the finding about a glycemic responsedifferent in sportsmen from that expected according to the studiescarried out in sedentary people.

From these studies about the action of DHA over the sports performanceand glycemia, it can be concluded the following:

1) It is been proved that the continuous ingestion of DHA for more than3 weeks produces an increase in the Plasma Total Antioxidant Capacity(PTAC) in a general and statistically significant way (p<0.05) in bothcompetitive and amateur cyclists. Also, the oxidative damage to lipidsis lower (p<0.05) (difference which is more important for trainedsportsmen than for amateur cyclists). Finally, it has been shown thatthe damage to DNA measured by urinary marker (8-oxodG) decreases aftertaking DHA for three weeks (p<0.035).

2) It has been proved that after 4 months of continuous ingestion ofDHA, the sports performance is higher (increase of charge and cardiacfrequency, as well as their percentage of VO₂max in the UV2). Also, astatistically significant normoglycemia in the effort trial for 90minutos at 75% of VO₂max carried out four months later consuming DHA hasbeen observed.

The association of both effects (an enhance in the sports performanceand normoglycemia during long period exercise) is a result which was notexpected nor known in the art.

Furthermore, it could be concluded that this association of effects isdesirable and could be ergogenical aids still not known.

Another object of the present invention is the use of docosahexaenoicacid for manufacturing a composition for enhancing sports performanceand maintaining blood glucose levels after physical exerciseadministered by any suitable means.

It should be considered that the European Union Scientific Committee onFood recommends the following components for a composition of beverageto be taken during a physical exercise (see,http://ec.europa.eu/food/fs/sc/scf/out64_en.pdf).

80 kcal/1000 ml Energy 350 kcal/1000 ml 20 mmol/l (460 mg/l) Na⁺ 50mmol/l (1150 mg/l) 200 mOsml/kg water Osmolarity 330 mOsml/kg water Atleast 75% of caloric energy must derive from carbohydrates with a highglycemic charge (glucose, maltodextrine, sucrose) Vitamine B1 0,2 mg/100mg carbohydrates

In this regard, the inclusion of carbohydrates is aimed to maintain theglycemia in order to avoid the fast consumption of muscular and hepaticglycogen. It should be considered the drawbacks of gastric emptyingdiminished due to the increase of osmolarity generating the presence ofconcentrations of carbohydrates, associated with the feeling of gastricfullness undesirably for a lot of sportsmen. Por consiguiente, preparinga beverage with a lower concentration of carbohydrates by adding DHAcould be an ergogenic advantage of undoubted interest in the sportsperformance.

Accordingly, another aspect of the present invention relates to apharmaceutical composition comprising DHA which can be used in the inthe food industry for the purpose of enriching food products (e.g. dairyproducts such as yoghurts, milk, etc) with a natural antioxidant agentsuch as DHA, or further, incorporated into a suitable administrationform selected from the group comprising a beverage in all itscharacteristics for before, during and after physical exercise;energy-giving bar; ergogenical bars; solids and preparations forprovisioning; dietetic supplement and polivitaminic preparation (in theform of, for example, capsules, tablets, pills, lyophilised form, or anysuitable mean of administration); ergogenical aids; textiles withnanocapsules for skin absorption and any other suitable mean ofadministration.

KEYS OF THE FIGURES

FIG. 1 Effect of DHA concentration in the Foreskin cells culture mediumon the intracellular generation of ROS. The cells were cultured in thepresence of a triglyceride with 70% by weight of DHA in relation to thetotal fatty acids for three days prior to the experiment. (A) ROSdetection was carried out with DHR 123 on cells treated with 40 or 60 mMAAPH for 180 min. The data show the mean of three independentexperiments. (B) The detection of ROS was carried out with CDCFDA oncells treated with the xanthine/xanthine oxidase system for 180 min. Byway of comparison, the data obtained with 100 μM Vitamin E (control) areincorporated. The data represent the mean of three independentexperiments.

FIG. 2 Comparative effect of the proportion of DHA of a triglyceride inthe Foreskin cells culture medium on the intracellular generation ofROS. (A) The cells were cultured in the presence of each triglyceridefor three days prior to the experiment. The concentration on the x-axisis the equivalent that would be obtained with a triglyceride having aDHA content of 70% by weight. The detection of ROS was carried out withDHR 123 on cells treated with 40 mM AAPH for 180 min. The data representthe mean of three independent experiments. (B) Representation of theantioxidant protection in relation to DHA concentration in the oil of20, 50 and 70%.

FIG. 3 Effect of DHA concentration on the production of TBARS inForeskin cells. The cells were cultured in the presence of atriglyceride with 70% by weight of DHA in relation to the total fattyacids for three days prior to the experiment at the concentrationindicated. The oxidative stress was induced with 40 mM AAPH for 6 h and24 h of latency. The data represent the mean of three independentexperiments.

FIG. 4 Effect of DHA concentration in the Foreskin cells culture mediumon the generation of superoxide anions. The cells were cultured in thepresence of a triglyceride with 70% by weight of DHA in relation to thetotal fatty acids for three days prior to the experiment. The detectionof superoxide anions was carried out by chemiluminiscence immediatelyfollowing oxidative induction of the cells with 40 mM AAPH and in someexperiments in the presence of 10 mM Tyron or of 0.1875 UA/μl ofexogenous SOD. The data are representative of three independentexperiments.

FIG. 5A Effect of DHA concentration in the Foreskin cells culture mediumon SOD activity. The cells were cultured in the presence of atriglyceride with 70% by weight of DHA in relation to the total fattyacids for three days prior to the experiment at DHA concentrations of0.5 (A), 5 (B) and 50 μM (C). The SOD activity was analysed indirectlyby analysing the decrease in the chemiluminiscence generated by theluminol as a consequence of the endogenous SOD activity. Oxidativeinduction was carried out with the 0.1 mM xanthine/0.005 U/ml xanthineoxidase system that immediately generates superoxide anions. The dataare representative of three independent experiments.

FIG. 5B Effect of DHA concentration in the Foreskin cells culture mediumon SOD activity. The cells were cultured in the presence of atriglyceride with 70% by weight of DHA in relation to the total fattyacids for three days prior to the experiment. The SOD activity wasevaluated on the non-induced cellular system or the system induced with40 mM AAPH. The data are representative of three independentexperiments.

FIG. 6 Effect of DHA concentration in the Foreskin cells culture mediumon GPx activity. The cells were cultured in the presence of atriglyceride with 70% by weight of DHA in relation to the total fattyacids for three days prior to the experiment. GPx activity was evaluatedon the non-induced cellular system or the system induced with 40 mMAAPH. The data are representative of three independent experiments.

FIG. 7 Effect of DHA concentration in culture medium of ARPE-19 cells onthe intracellular generation of ROS. The cells were cultured in thepresence of a triglyceride with 70% by weight of DHA in relation to thetotal fatty acids for three days prior to the experiment. (A) Thedetection of ROS was carried out with DHR 123 (A) or with CDCFDA (B) oncells treated with 40 or 60 mM of AAPH for 180 min. The data representthe mean of three independent experiments.

FIG. 8 Comparative effect of DHA concentration of a triglyceride in theculture medium of ARPE-19 cells on the intracellular generation of ROS.The cells were cultured in the presence of each triglyceride for threedays prior to the experiment. (A) The concentration on the x-axis is theequivalent that would be obtained with triglyceride having a DHAproportion of 70% by weight. The detection of ROS was carried out withDHR 123 on cells treated with 40 mM de AAPH for 180 min. The datarepresent the mean of three independent experiments. (B) Representationof the antioxidant protection in relation to DHA concentration in theoil of 20, 50 and 70%.

FIG. 9 Effect of DHA concentration on the production of TBARS in ARPE-19cells. The cells were cultured in the presence of a triglyceride with70% by weight of DHA in relation to the total fatty acids for three daysprior to the experiment at the indicated concentration. The oxidativestress was induced with 40 mM AAPH for 6 h and 24 h of latency. The datarepresent the mean of three independent experiments.

FIG. 10 Effect of DHA concentration in the ARPE-19 cells culture mediumon the generation of superoxide anions. The cells were cultured in thepresence of a triglyceride with 70% by weight of DHA in relation to thetotal fatty acids for three days prior to the experiment. The detectionof superoxide anions was carried out by chemiluminiscence immediatelyfollowing oxidative induction of the cells with AAPH 40 mM. The data arerepresentative of three independent experiments.

FIG. 11 Effect of DHA concentration in the ARPE-19 cells culture mediumon GPx activity. The cells were cultured in the presence of atriglyceride with 70% by weight of DHA in relation to the total fattyacids for three days prior to the experiment. GPx activity was evaluatedon the non-induced cellular system or the cellular system induced with40 mM AAPH. The data are representative of three independentexperiments.

FIG. 12 Effect of DHA concentration in the ARPE-19 cells culture mediumon SOD activity. The cells were cultured in the presence of atriglyceride with 70% by weight of DHA in relation to the total fattyacids for three days prior to the experiment. SOD activity was evaluatedon the non-induced cellular system or the cellular system induced with40 mM AAPH. The data are representative of three independentexperiments.

FIG. 13 Effect of DHA concentration obtained by chemical synthesis (Aand C) or enzymatic synthesis (B and D) on the percentage of cellularprotection versus oxidative stress in ARPE-19 cells (A and B) orForeskin cells (C and D).

FIG. 14 Influence of purification degree of the oil obtained by chemicalsynthesis on the percentage of cellular protection versus oxidativestress induced by DHA in ARPE-19 cells.

FIG. 15 Influence of chemical structure on the percentage of cellularprotection versus oxidative stress induced by DHA in ARPE-19 cells.

FIG. 16 Effect of DHA concentration on intracellular concentration ofglutation in ARPE-19 cells. Influence of the presence of BSO.

FIG. 17 Influence of glutation de novo synthesis on the percentage ofcellular protection versus oxidative stress induced by DHA in ARPE-19cells.

FIG. 18 Effect of DHA concentration on intracellular concentration ofglutation in Foreskin cells. Influence of the presence of BSO.

FIG. 19 Influence of purification degree of the oil obtained by chemicalsynthesis on the percentage of cellular protection versus oxidativestress induced by EPA in ARPE-19 cells. Comparative study with DHA.

FIG. 20 Effect of EPA concentration on the percentage of cellularprotection versus oxidative stress in Foreskin cells. Comparative studywith DHA.

FIG. 21 Effect of EPA concentration on intracellular concentration ofglutation in Foreskin cells. Influence of the presence of BSO.

FIG. 22 is a comparative bar graphic showing the effect of the DHApercentage in a structured and non-structured triglyceride at differentdosages in respect with the percentage of cell protection.

Said FIG. 22 shows the surprising results of the object of the presentaddition when comparing a non-structured glyceride chemical structure(triglyceride) with the same structure wherein sn-1 and sn-3 positionshave been replaced with caprylic acid (structured), both from anenzymatic source with two starting levels in content of DHA of 20 and70%.

From the figure, it can be observed that at the same concentration, thepercentage of protection of the docosahexaenoic acid incorporated intothe sn-2 position of a glyceride (structured), in particular, atriglyceride, shows an efficiency which is approximately 3 times higherthan that of a glyceride containing non-structured DHA.

In such a FIG. 22, the protection percentage indicates the relationshipbetween the difference in the intracellular concentration of reactiveoxygen species of control cells and those treated with DHA in respectwith the control cells, both subjected to the same oxidative stressexpressed in percentage. In other words, the existence of a protectionpercentage indicates in the treated cells a significant statisticallyless intracellular generation of reactive oxygen species in respect withthe control.

FIG. 23 is a comparative graphic showing the average length of thetelomere in human fibroblasts cultured under oxidative stress with orwithout DHA incorporated vs. the pass number of cellular populations.

Said FIG. 23 shows the surprising results of the object of the presentaddition at observing that in presence of DHA under oxidative stressconditions, the telomere shortening index is lower in respect to thecontrol or without DHA.

FIG. 24 is a graphic representing the absolute oxygen consumption in the“ventilatory threshold 2” (UV2) for competing, non-competing and allcyclists at basal level and after 4 months taking DHA.

FIG. 25 is a graphic representing the cardiac frequency in UV2 forcompeting, non-competing and all cyclists at basal level and after 4months taking DHA.

FIG. 26 is a graphic representing the time needed to reach the UV2 forcompeting, non-competing and all cyclists at basal level and after 4months taking DHA.

FIG. 27 is a graphic representing the cardiac frequency during theconsumption of 2000 ml/min O₂ in the ventilatory threshold forcompeting, non-competing and all cyclists at basal level and after 4months taking DHA.

FIG. 28 is a graphic representing the plasma total antioxidant capacityfor competing, non-competing and all sportsmen at basal level and after3 weeks taking DHA. In each case, there is shown the antioxidantcapacity before (left bar) and the antioxidant capacity after (rightbar) the effort trial.

FIG. 29 is a graphic representing the oxidative damage to plasmaticlipids according to MDA concentration for competing, non-competing andall sportsmen at basal level and after 3 weeks taking DHA. In each case,there is shown the oxidative damage before (left bar) and the oxidativedamage after (right bar) the effort trial.

FIG. 30 is a graphic representing the oxidative damage to DNA using theoxidative stress biomarker 8-oxodG for competing, non-competing and allsportsmen at basal level and after 3 weeks taking DHA. In each case,there is shown the oxidative damage before (left bar) and the oxidativedamage after (right bar) the effort trial.

FIG. 31 is a graphic representing the glycemia in competing sportsmenduring a physical effort who did not take DHA or did it for 3 weeks or 4months.

FIG. 32 is a graphic representing the glycemia in non-competingsportsmen during a physical effort who did not take DHA or did it for 3weeks or 4 months.

FIG. 33 is a graphic representing the glycemia in competing andnon-competing sportsmen during a physical effort who did not take DHA ordid it for 3 weeks or 4 months.

The following examples are included by way of illustrative andnon-limitative examples of the invention.

EXAMPLES Materials and Methods for Evaluating Antioxidant Activity CellCultures

The cellular models used were Foreskin cells (undifferentiated epidermalfibroblasts, CRL-2076) and ARPE-19 cells (retina pigmentary epithelialcells, CRL-2302) obtained from the American Type Culture Collection. Thecell cultures were kept in suitable growth conditions of temperature(37° C.), CO₂ concentration (5%) and humidity (95%) in an incubatorspecially designed for this purpose. The ARPE-19 cells were maintainedin growth up to confluence of 0.3×10⁴ cells/cm² in culture flasks withDMEM-F12 medium (Biological Industries) supplemented with 10% bovinefoetal serum, penicillin antibiotics (100 U/mL), streptomycin (100μg/mL) and glutamine (Biological Industries). The CRL-2076 fibroblastswere kept growing in culture flasks in Iscove's modified Dulbecco'smedium (Biological Industries) supplemented with 10% bovine foetalserum, penicillin antibiotics (100 U/mL), streptomycin (100 μg/mL) andglutamine (Biological Industries). The cells were transferred foradherence to the substrate 24 h at 37° C. from the 75 ml flasks to 6, 12or 96-well plates in order to be able to carry out the experiment (10⁶cells/mL).

Integration of the DHA into the Cells

DHA-TG was added at various concentrations (0.5-50 μM) starting with theDHA-TG enriched with 20, 50 and 70% (oil density 0.92 g/mL), made bydissolving the oil in ethanol for the stock solution (1:100) andpreparing the working solutions in a culture medium prepared with serum.The cells were cultured with supplemented DHA-TG medium for 3 days at37° C.

Inducing Oxidative Stress

Various inducer cells were used to stress the cells oxidatively:

a) xanthine/xanthine oxidase system 0.8 mM/10⁻² U/mL that catalyses theoxidation of hypoxanthine and xanthine to uric acid, with reduction ofO2 to O.⁻² and H₂O₂.

b) 2,2′-azobis-(2-amidinopropane)dihydrochloride (AAPH) 1-100 mM widelyused as a hydrophilic initiator of free radicals by inducing lipidic andprotein peroxidation. The AAPH oxidises the DNA, the proteins and thelipids through the action of the formed peroxil radicals. It furtheracts on the endogenous defence system, since it deactivates the keyenzyme, the SOD, thereby losing the protective capacity of the CAT andthe GPx.

Generation of Reactive Oxygen Species (ROS)

The ROS level was measured in primary cultures of human skin CRL-2076fibroblasts and in ARPE-19 retinal epithelial cells by employing thefluorimetric technique using dihydrorodamine 123 (DHR123, MolecularProbes) and 2,7-dichlorofluorescein diacetate (H₂DCFDA, MolecularProbes) as fluorescent probes in a continuous system measuring every 30min until 180 minutes. In both cases, this is an unspecific measurementof ROS generation. The fluorescent probes were added to the cells (1×10⁶cells/mL) at a final concentration of 10 μM. The fluorescence of theoxidised probes (2,7-dichlorofluorescein and rodamine 123) was measuredin a Mithras fluorescence reader at an excitation wavelength of 488 nmand an emission wavelength of 525 nm in function of the time. Thefluorescence obtained is modulated with the cellular viabilitydeterminations by the MTT spectrophotometric technique outlined below.

Cellular Viability

Cellular viability studies were carried out in order to evaluate thecytotoxic effect of various samples. This method consists of adding theMTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoyl bromide,Sigma), soluble in aqueous medium, to the incubation medium. The viablecells metabolise this compound and it is converted into formazan salt.This salt is a colorimetric compound insoluble in aqueous medium,soluble in DMSO and usable for measuring cellular viability. The methodconsists of adding 20 μl per well of a 7.5 mg/ml (in excess) MTTsolution. This is incubated for one hour at 37° C. so that the viablecells metabolise the compound and produce the formazan salt, while thenon-viable ones do not. After incubating for one hour the cells areprecipitated and 100 μl of DMSO added, which will dissolve the formazansalt. Finally, the absorbance at 550 nm is read on a plate reader. Theviability results are expressed as an optical density percentage inrelation to the controls, taking the latter to have 100% viability.Cellular viability curves were drawn up on 96-well plates by sowingabout 20,000 cells per well (following analysis of the suitable numberof cells in function of their growth ratio) with an approximate volumeof 200 μl of medium per well. The study of the efficiency of the productis carried out after exposing the cells to the product for 72 h in asufficiently wide range of concentrations to find the value of IC₅₀. Theexperimental results are adjusted to the Hill equation using the SigmaPlot 8.0 to determine the IC₅₀, defined as the DHA concentrationnecessary to reduce the viability of the culture to 50% in relation tothe control.

Determination of Proteins

The determination is based on colorimetric detection and totalquantification of the proteins with an optimised dizinconinic acidformulation that allows proteins to be measured in diluted samples in aconcentration range of 0.5-20 μg/ml. The method uses a detector forCu⁺¹, which is reduced by the proteins in alkaline medium to Cu⁺². Thepurple reaction product is formed by chelation of two molecules of BCAwith the cuprous ion. The water-soluble complex absorbs at 562 nm. Bymeans of a calibration curve an equation can be obtained, with theresults expressed in μg/mL of proteins. The commercial kit used is theMicroBCA from Pierce (No. 23235).

Direct Analysis of ROS Generation Measurement of Generation of LipidicHydroperoxides

The measurement of malonildialdehyde (MDA) on cell lysates was used as amarker of lipidic peroxidation by UV-Vis spectrophotometry. The MDA andthe 4-hydroxyalkenals (HAE) are products derived from the peroxidationof polyunsaturated fatty acids and related esters. Direct measurementsof these aldehydes constitutes a convenient index of lipidicperoxidation. A chromogenic reagent (N-methyl-2-phenyl-indole inacetonitrile) which reacts with the MDA at 45° C. was employed, usingthe commercial lipidic peroxidation kit from Calbiochem (No. 437634).The condensation of one molecule of MDA with two molecules of thechromogenic reagent gives a stable chromophore with maximum absorbanceat 586 nm, with the detection limit being 0.1 μM. The induction wascarried out for 6 h with 40 mM AAPH and 24 hours of latency. The cells(10⁷ cells/mL) were lysed by means of cycles of freezing and thawing inliquid N2. The samples were fractionated in order to measure MDA andprotein. The results were expressed in μM of MDA/mg of protein.

Measurement of Generation of Superoxide Anion

Direct measurement of the superoxide anion was carried out by means ofthe chemiluminescence technique on microplate measured by luminol(Calbiochem, No. 574590). Chemiluminiscence for detecting the superoxideanion is a technique used due to its potential for gaining access to allthe intracellular sites of superoxide generation, due to the highspecificity of the reaction with luminol, the minimal intracellulartoxicity and the increased sensitivity in relation to other chemicaltechniques. It is based on the superoxide anion oxidising luminol in areaction that produces photons of light which are quickly measured on astandard illuminometer. In our tests we used a chemiluminescence readeron microplate from ELISA, MITHRAS and furthermore, given the shorthalf-life of the radical, an enhancer was used to increase thesensitivity of the test and amplify the response. This reagent can beused on living cells, since it is not toxic and does not denature thesubcellular system components. The capacity for inhibiting theproduction of superoxide anion was also investigated using a specificsuperoxide anion sequestering agent, Tyron (4,5-dihydroxy-1,3-benzenedisulphonic acid, Sigma) frequently used for in vitro blocking assays onROS production, being permeable to the cell membrane and superoxidedismutase (SOD, Sigma) was used as an enzyme blocker, constituting afirst-line enzyme in the endogenous antioxidant defence. Thechemiluminiscence measurement in the cells submitted to the AAPHoxidative stress inducing treatment was analysed every 60 seconds for atotal time of 4100 seconds, at a frequency of 120 sec/cycle. The resultswere expressed in UA of chemiluminiscence/mg protein.

Determining Antioxidant Enzyme Activity Measuring Glutation Peroxidase(GPx) Activity

GPx catalyses the reduction of hydroperoxides to reduced glutation, thefunction being to protect the cell from oxidative damage. It usesglutation as last electron donor to regenerate the reduced form ofselenocysteine. The indirect measurement of GPx is obtained by coupledreaction with glutation reductase. The oxidated glutation (GSSG)produced by the reaction with the hydroperoxides by action of the GPx isrecycled to its reduced state by the glutation reductase using NADPH ascoenzyme. Oxidation from NADPH to NADP⁺ is accompanied by reduction ofits absorbance at 340 nm. The rate of reduction of the absorbance at 340nm is directly proportional to the GPx activity of the sample. The ELISAmicroplate spectrophotometric kit from Cayman (No. 703102) was used fordetecting the GPx in cell lysates of primary cultures. The cells werecultured by adherence to the substrate for 24 h at 37° C. The celllysate was obtained by sonication in Tris 50 mM pH 7.5, EDTA 5 mM andDTT 1 mM. The activity of the GPx is obtained by determining the changeof A₃₄₀ nm/min (ΔA340), expressed as nanomoles NADPH/min/mg of proteinfrom the sample.

Measuring the Superoxide Dismutase Activity (SOD)

This chemiluminescence methodology is based on the analysis of SODactivity in the supernatant cellular in relation to a positive controlof SOD (Calbiochem No. 574590). The presence of SOD in the xanthineoxidase-xanthine-luminol system leads to a reduction of thechemiluminiscence produced as a reduction of dismutation of thesuperoxide anion proportional to the SOD activity. The analysis iscarried out on a MITHRAS illuminometer at intervals of 50 msec up to afinal reaction time of 520 sec.

The superoxide dismutase activity (SOD) in cellular lysates by means ofthe reaction using tetrazolium salts for detecting superoxide radicalsgenerated by xanthine oxidase/hypoxanthine system has been alsodetermined. An spectrophotometric method is used on a microplate formeasuring the 3 types of SOD (Cu—Zn-SOD; Mn-SOD and Fe-SOD), that iscytosolic and mitochondrial). One unit of SOD is defined as the quantityof enzyme required for dismuting 50% of the generated superoxide anion.In order to detect SOD in cellular lysates from primary cultures aCayman kit (N. 706002) was used following the protocol optimized by themanufacturer. The dynamic range of the assay is 0.025-0.25 SOD units/ml.

Determination of Intracellular Endogenous Antioxidant ConcentrationMeasuring the Reduced Glutation Intracellular Concentration (GSH)

Direct kinetic assay for measuring reduced glutation (GSH) in cellularlysates. Glutation can be found inside the cells mainly in the reducedform (90-95% of total glutation), being the main antioxidant in tissues.Its role is detoxifying xenobiotics and removing hydroperoxides so as tokeep the cellular redox state. The technique employed measures the totalglutation (GSSG+GSH) in a biological sample (cellular lysate) previouslydeproteinized with sulphosalicylic acid (Sigma-Aldrich CS0260 kit). GSHcauses a continuous reduction from 5,5′-dithiobis(2-nitrobenzoic) acid(DTNB) to 5-thio(2-nitrobenzoic acid (TNB) and the GSSG formed isrecycled by glutation reductase and NADPH. TNB is spectrophometricallymeasured at 412 nm. Buthionine sulfoximine (BSO) specifically inhibitinggamma-glutamylcysteine synthetase was used as a synthesis inhibitor.

Evaluation of the Anti-Oxidant Activity of DHA in a Human Skin Model

In this in vitro assay Foreskin cells (undifferentiated epidermalfibroblasts, ATCC CRL-2076) were used as cellular model, being asuitable cellular type due to their good in vitro response to variousoxidant inducers, in addition to being a primary culture with normalnutritional requirements and culture conditions, thus constituting agood in vitro model extrapolable to the in vivo response, for apotential cosmetic application of the DHA.

Results

The conditions were laid down initially to achieve an active cellularmodel under all study conditions. This means that the results obtainedrefer to metabolically active cells. Prior studies had already shownthat in Foreskin cells concentrations of less than 1000 μM of DHA didnot affect cellular viability in studies at 3 days. Neither was cellularviability affected for the studies of oxidative stress with thexanthine/xanthine oxidase system or with AAPH. It has also been shownthat the incorporation of DHA up to a concentration of 50 μM in aculture of Foreskin cells for 3 days does not significantly increase thecellular oxidative level measured as cellular fluorescence associatedwith two probes, dihydrorodamine (DHR 123) and 2,7-dichlorofluorescein(H2DCFDA), more specific for superoxide anion and for the detection ofhydroperoxides, respectively. Once these conditions had beenestablished, the general antioxidant capacity of the DHA incorporatedinto the membrane of the Foreskin cells was evaluated against oxidativestress induced by xanthine/xanthine oxidase or by AAPH.

When inducing a moderate oxidative stress with 40 mM AAPH and usingDHR123 as ROS detector, the DHA shows an inhibiting effect on thegeneration of the reactive oxygen species, both at the concentration of0.5 μM (59% protection) and at 5 μM (33% protection), showing a lowereffect at 10 μM (26% protection) or no effect at 50 μM of DHA (FIG. 1A).When the cells are submitted to severe induction with 60 mM AAPH, theDHA shows a protective effect against the generation of ROS, both at 0.5μM concentration (40% protection) and 5 μM (29% protection), but losingit at higher concentrations of DHA (FIG. 1A).

We might also note the protection that 0.5 μM DHA exercises against theoxidative stress induced by the xanthine/xanthine oxidase (FIG. 1B),which shows a sequestering effect on the oxygen reactive species, bothsuperoxide anion and hydroperoxides generated in the oxidative process.Comparing the antioxidant capacity in relation to a lipophilicantioxidant such as vitamin E (FIG. 1B), we observe that they exercisesimilar protection kinetics (with DHA inhibiting cellular oxidation by33.46% and vitamin E by 30%).

The protection kinetic response of the DHA always presents a maximumantioxidant effect between 60-120 minutes after carrying out theinduction, thus denoting a saturation in the hydroperoxides andsuperoxide anion sequestering capacity of the DHA. The antioxidantbehaviour is critically dose-dependent, since increasing theconcentration thereof leads to a loss of ROS sequestering capacity, withthe 0.5 μM concentration having the most effective antioxidant capacity.In this regard, another critical parameter in terms of optimising theefficiency of the system is the proportion of DHA in relation to totalfatty acids. As shown in FIG. 2, at identical concentrations oftriglyceride, a reduction of the proportion of DHA to 50 or 20%drastically reduces the cellular antioxidant capacity, and it reverts tobeing pro-oxidant at low or moderate concentrations. These resultsappear to indicate that the cellular antioxidant effect of the DHA doesnot depend exclusively on the concentration thereof, but also it is adecisive factor its molecular localisation, in this case itsdistribution in the structure of the triglyceride.

As regards specific inhibition of ROS production, we analysed thegeneration of lipidic peroxides (TBARS) and of superoxide anions. Theresults obtained showed that the cells treated with AAPH generated ahigher concentration of substances reactive to thiobarbituric acid(TBARS) when compared with the non-induced cells, expressed as μM ofMDA/mg of proteins (FIG. 3). As expected, incorporation of DHA into themembrane of the Foreskin cells slightly increased the basal cellularlipidic peroxidation in dose-dependent form (0.5, 5 and 50 μM) (FIG. 3).In the cells submitted to oxidative induction with 40 mM AAPH, the DHApresents an antioxidant activity protecting the fibroblasts fromgenerating membrane hydrolipidic peroxides, its action being of theinverse concentration-dependent type. The protection with DHA was 87%for 0.5 μM DHA, 85% for 5 μM and 48% for 50 μM DHA-TG (FIG. 3).

Generation of the superoxide anion was then analysed. Foreskin cellssubmitted to an oxidative stress with 40 mM AAPH generated a superoxideanion production 2.5 times greater than the non-induced cells, whichmaintained a constant superoxide anion level (FIG. 4). In the absence ofoxidative induction the cells with integrated DHA do not show a higherlevel of intercellular superoxide anion in relation to control (FIG. 4).Under oxidative stress conditions (FIG. 4) the DHA inhibits generationof the superoxide anion by 16.5% at a concentration of 0.5 μM, by 10% ata concentration of 5 μM and by 9% at a concentration of 50 μM. Thespecificity of the method was confirmed by the addition of Tyron(4,5-dihydroxy-1,3-benzene disulphonic acid, a compound which ispermeable to the cellular membrane that operates as a highly specificsequestering agent of intracellular superoxide anion) or ofextracellular SOD (first-line enzyme blocker in the endogenousantioxidant defence via dismutation of the intracellular superoxideanion). The production of the superoxide anion in cells stressed withAAPH, with or without DHA previously integrated, in the presence ofexogenous SOD or of Tyron, was totally inhibited and achieved basalvalues (FIG. 4).

Finally, we analysed if the DHA underwent its antioxidant activity bymodifying the activity of the first-line cellular antioxidant enzymes.The activity of the SOD and of the GPx in Foreskin cells with or withoutintegrated DHA was analysed. In the first case, the xanthine/xanthineoxidase system was used as instantaneous generator of superoxide anions(total measuring time 520 sec., measuring every 50 msec.). The resultsobtained showed a good oxidative induction with rapid kinetics, withdirect observation of dismutation and non-production of superoxideanion. The maximum chemiluminiscence achieved after 15 seconds fromoxidative induction was interpreted as an indirect and qualitativemeasurement of SOD activity (FIG. 5A). Without DHA integrated, values of310 U.A. chemiluminiscence/10⁶ cells were achieved, falling to 150 U.A.chemiluminiscence/10⁶ cells in a system pre-incubated with DHA 0.5 μM(52% antioxidant protection) (FIG. 5A). The antioxidant efficiency wasmaintained at 52% and 42% protection in cells treated with 5 and 50 μMof DHA, respectively (FIG. 5A). Furthermore, knowing that AAPH oxidisesthe DNA, the proteins and the lipids by diffusion of the generatedperoxil radicals, the DHA as antioxidant may prevent deactivation of theSOD entrusted with dismutation of the superoxide anion, maintaining inthe cell the endogenous antioxidant defence of the catalase and theglutation peroxidase. This aspect is confirmed in FIG. 5B, wherein SODactivity is shown not to be increased in basal state with DHA beingpresent (−10/−15%), but loss of SOD activity inherent to the oxidativestress process is inhibited with DHA being present keeping or evenincreasing SOD activity (10/20%). As for GPx activity (FIG. 6), this isfound to be increased in cellular basal state at modest concentrationsof DHA (up to 17% at 5 μM), but falls off at high concentrations (−20%at 50 μM). This behaviour is maintained intact in an oxidative stressstate (FIG. 6). These results suggest that the DHA collaborates with theendogenous antioxidant defence system as relates to dismutation of thesuperoxide anion by generating SOD over the entire range ofconcentrations tested, and is also capable of controlling the generationof hydroperoxides at moderate concentrations, since it increases GPxactivity.

Evaluation of the Antioxidant Activity of DHA in a Retina Cellular Model

In this in vitro study the cellular model was based on ARPE-19 cells(pigmentary retinal epithelial cells, ATCC CRL-2302), being a suitablecellular type due to their good in vitro response to various oxidantinducers, as well as being a primary culture with normal nutritionalrequirements and culture conditions. It also constitutes a good ocularmodel, since it keeps the biological and functional properties of theretinal pigmentary epithelial cells.

Results

The assay carried out with this cellular line is similar to thatdescribed for the Foreskin cells in the preceding section. The basicrequirements were the same in relation to keeping cellular viabilityunder all working conditions (effect of the DHA, of oxidative stress).Neither did incorporation of DHA at the doses analysed involved anysignificant alteration in the basal cellular oxidative state.

On inducing a moderate oxidative stress with 40 mM AAPH and using DHR123as ROS detector, the DHA shows an inhibiting effect on the generation ofthe reactive oxygen species, at the concentrations of 0.5 μM (43%protection) and 5 μM (32% protection), but with a lower effect at 50 μM(4% protection) of DHA (FIG. 7A). When the cells are submitted to severeinduction with 60 mM AAPH, the DHA shows a protective effect against ROSgeneration, at the 0.5 μM concentration (13% protection) and lower athigher concentrations of DHA (FIG. 7A). These results are similar tothose obtained with the Foreskin cells, although one notabledifferential effect is the lower protection observed against a severeoxidative induction. By using for the ROS detection, the CDCFDA morespecific to peroxides, it is also revealed the protection that the DHAexercises against the oxidative stress induced by AAPH (FIG. 7B).

The protection kinetics of the DHA also always presents a maximumantioxidant effect 60-120 minutes after carrying out the induction,denoting a saturation in the DHA's hydroperoxides and superoxide anionsequestering capacity. Quantitatively, the antioxidant capacity iscritically dose-dependent, since when DHA concentration is increasedthere is a loss of ROS sequestering capacity, with the 0.5 μMconcentration being the most effective in its antioxidant capacity(FIGS. 7A and 7B). In this respect, another critical parameter in termsof optimising the efficiency of the system is the ratio of DHA to totalfatty acids. Reducing the proportion of DHA in relation to total fattyacids from 70% to 50-20% significantly and non-proportionally reducesits cellular antioxidant capacity at the optimum concentrations (0.5-5μM), rendering it equal to the high concentrations (FIGS. 8A and 8B),though unlike to the Foreskin cells at no proportion does the DHA becomepro-oxidant. These results confirm that the cellular antioxidant effectof the DHA does not depend exclusively on its concentration, but also adecisive factor is its molecular localisation, in this case itsdistribution in the structure of the triglyceride.

As regards specific inhibition of ROS production, the generation oflipidic peroxides (TBARS) (FIG. 9) and of superoxide anions (FIG. 10)were analysed. The results obtained are very similar to those obtainedwith the Foreskin cells. The cells treated with AAPH generate a higherconcentration of substances reactive to thiobarbituric acid (TBARS) andof superoxide anions in relation to the non-induced cells. Theincorporation of DHA into the membrane of the ARPE-19 cells slightly anddose-dependently (0.5, 5 and 50 μM) increases the cellular basal lipidicperoxidation, though in the cells submitted to oxidative induction, theDHA presents a cellular antioxidant activity inhibiting them fromgenerating membrane lipidic hydroperoxides in an inverse ratio to theirconcentration. The protection with DHA was 64% for 0.5 μM DHA, 58% for 5μM and 42% for 50 μM DHA (FIG. 9). Generation of the superoxide anionwas then analysed. In the absence of oxidative induction, the cells withintegrated DHA do not present a higher level of intracellular superoxideanion in relation to the control (FIG. 10A). An oxidative stress with 40mM AAPH generates a superoxide anion production that is partiallyinhibited by the DHA (20-16% at concentrations of 0.5-50 μM). Thisinhibition is in concordance with SOD activity with DHA being present(FIG. 10B). SOD activity is not found to be increased in basal statewith DHA being present (−10/15%), but as in Foreskin cells, loss of SODactivity inherent to the oxidative stress process is inhibited with DHAbeing present keeping basal SOD activity.

Finally, an analysis was carried out to find whether the DHA altered theactivity of the GPx enzyme as first-line cellular antioxidant (FIG. 11).The GPx activity is increased in cellular basal state at all theconcentrations of DHA tested (12-40%), and this behaviour is maintainedintact in oxidative induction state, which also presents a 2.5 timeshigher GPx activity (FIG. 11). As in the case of the Foreskin cells,these results suggest that the DHA exercises part of its antioxidanteffect by modulating the activity of the endogenous cellular enzymesystem antioxidant defence.

Influence of Synthesis Method in the Antioxidant Activity of DHAIncorporated into a Triglyceride

In the present in vitro assay, ARPE-19 cells (retina pigmentaryepithelial cells, ATCC CRL-2302) and Foreskin cells (undifferentiatedepidermal fibroblasts, ATCC CRL-2076) were used as cellular model, beingsuitable cellular lines due to their good in vitro response to variousoxidant inducers. Tuna oil triglycerides (DHA20%-TG, 20% molar in DHA)or oil derivatives enriched with 50 or 70% molar in DHA (DHA50%-TG andDHA70%-TG) obtained by chemical methods (CHEM) or enymatic methodes(ENZ) were used an active ingredient.

Results

When inducing a moderate oxidative stress with 40 mM AAPH in ARPE-19cells and using DHR123 or H2DCFDA as ROS intracellular detectors, thenatural DHA (DHA20%-TG) and that incorporated into a chemically obtainedtriglyceride (DHA50%-TG-CHEM and DHA70%-TG-CHEM) shows an inhibitoryeffect in the generation of the reactive oxygen species, both 0.5 μM and5 μM concentration, showing a lower effect at 50 μM (FIG. 13A). Thiseffect depends on the content of DHA, beingDHA70%-TG-CHEM >DHA50%-TG-CHEM >DHA20%-TG. At the same concentrations(0.5, 5 and 50 μM), enzimatically obtained oils show a higher activityat all DHA contents (DHA70%-TG-ENZ and DHA50%-TG-ENZ) (FIG. 13B). In asimilar study with Foreskin cells the results were even more surprising.The prooxidative activity shown with DHA70%-TG-CHEM and DHA50%-TG-CHEMat high dose (FIG. 13C) becomes antioxidative at all concentrations withoils with enzymatic origin (DHA70%-TG-ENZ and DHA50%-TG-ENZ) (FIG. 13D).The removal of intrinsic polymers of oils obtained chemically by meansof chromatographic methodes (DHA70%-Tg-BPM) causes a decrease evengreater of antioxidative activity in aRPE-19 cells, becomingprooxidative at high concentrations (5 and 50 μM) (FIG. 14). Theantioxidative activity of DHA incorporated into a triglyceride obtainedby enzymatic synthesis is also higher (at least twice) than that shownby Dha incorporated into other chemical structures such as ethyl esters,free fatty acid o fatty acid linked to serum albumin (FIG. 15).

The cellular antioxidative activity shown with the incorporation of DHAis related to all the aspects previously considered such as maintainingSOD and GPX enzymatic activities, but also to an increase in glutationintracellular concentration (GSH). In ARPE-19 cells (FIG. 16), DHAinduces an increase in the GSH intracellular concentration directlyrelated to GSH de novo synthesis since the addition of BSO (specificinhibitor of GSH synthesis) eliminates the protective effect of DHA(FIG. 17) in a direct relation with a decrease in the GSH intracellularconcentration (FIG. 15). A similar behaviour is shown for Foreskin cells(FIG. 18).

The improvement obtained in the antioxidative activity of DHA by anenzymatic synthesis is also applicable to another omega-3 fatty acidsuch as ecosapentaenoic acid (EPA). In a study with ARPE-19 cells, EPAobtained enzimatically (EPA70%-TG-ENZ) are shown to have anantioxidative activity, though very lower to that observed with DHA(DHA70%-TG-ENZ), whereas EPA obtained chemically and free of polymers(EPA-70%-TG-BPM) is shown to be very prooxidative (FIG. 19).Furthermore, EPA (EPA70%-Tg-ENZ) obtained enzimatically shows inForeskin cells a remarkable antioxidative activity even higher than thatfor DHA (DHA70%-TG-ENZ) (FIG. 20), related to, just like for DHA, theincrease of GSH intracellular concentration (FIG. 21).

Evaluating the Antioxidant Activity of the Dha Incorporated into aStructured Triglyceride in a Retina Cellular Model

In this in vitro assay ARPE-19 cells (retina pigmentary epithelialcells, ATCC CRL-2302) were used as cellular model, being a suitablecellular type due to their good in vitro response to various oxidantinducers, in addition to being a primary culture with normal nutritionalrequirements and culture conditions. Furthermore, it is a good ocularmodel since it keeps the biological and functional properties of theretina pigmentary epithelial cells. As an active ingredient there hasbeen used structured triglycerides derived from tuna oil (DHA20%-TG, 20%molar in DHA) or oil enriched with 70% DHA (DHA70%-TG, 70% molar inDHA), wherein through enzymatic methods the fatty acids in sn-1 and sn-3positions have been replaced with octanoic acid. In these new compounds,the molar content of DHA is 7% in the DHA20%-TG and 22% in DHA70%-TG.

Results (See FIG. 22)

When inducing a moderate oxidative stress with 40 mM AAPH and usingDHR123 as ROS detector, the DHA incorporated into a normal triglyceride(DHA20%-TG and DHA70%-TG) shows an inhibitory effect in the generationof the reactive oxygen species, both 0.5 μM and 5 μM concentration,showing a lower effect at 50 μM (FIG. 22). This effect depends on thecontent of DHA, being DHA70%-TG >DHA20%-TG. At the same concentrations,the structured oils, with a real DHA concentration 2-3 times lower, showthe same activity (for 0.5 M concentration) or higher (for 5 μM and 50μM concentrations) in the case of DHA20%-TG. In the case of DHA70%-TG,the efficacy of the structured triglyceride is slightly lower thanoptimum concentrations (0.5 μM and 5 μM), but the behaviour at highconcentrations is inverted (50 μM) showing in general a more stable andless dose-dependent behaviour.

Evaluating the Dha Activity as a Protective Agent of the Length of aTelomere Associated to the Age in a Human Skin Model

In this in vitro assay Foreskin cells (undifferentiated epidermalfibroblasts, ATCC CRL-2076) were used as cellular model, being asuitable cellular type due to their good in vitro response to variousoxidant inducers, in addition to being a primary culture with normalnutritional requirements and culture conditions, thus constituting agood in vitro model extrapolable to the in vivo response, for apotential cosmetic application of the DHA.

Methodology Cell Cultures

The cellular models used were Foreskin cells (undifferentiated epidermalfibroblasts, CRL-2076) obtained from the American Type CultureCollection. The cell cultures were kept in suitable growth conditions oftemperature (37° C.), CO2 concentration (5%) and humidity (95%) in anincubator specially designed for this purpose. The CRL-2076 fibroblastswere kept growing in culture flasks in Iscove's modified Dulbecco'smedium (Biological Industries) supplemented with 10% bovine foetalserum, penicillin antibiotics (100 U/mL), streptomycin (100 μg/mL) andglutamine (Biological Industries).

Integration of the DHA into the Cells

Enzymatically synthesized DHA-TG 70% was added at a 0, 5 μMconcentration, made by dissolving the oil in ethanol for the stocksolution (1:100) and preparing the working solutions in a culture mediumprepared with serum. The cells were cultured with supplemented DHA-TGmedium for 3 days at 37° C.

Induction of Oxidative Stress

2,2′-azobis-(2-amidinopropane)dihydrochloride (AAPH) was used to stressthe cells oxidatively at a concentration of 40 mM, widely used as ahydrophilic initiator of free radicals by inducing lipidic and proteinperoxidation. The AAPH oxidises the DNA, the proteins and the lipidsthrough the action of the formed peroxil radicals. It further acts onthe endogenous defence system, since it deactivates the key enzyme, theSOD, thereby losing the protective capacity of the CAT and the GPx.

Measurement of the Length of the Telomere

The telomeric regions constituted by high repetitive DNA can beevaluated by in situ hibridation techniques. The in situ hibridationmethod with fluorescence (FISH) using complementary probes to thetelomeric sequences allowed to detect the presence or absence oftelomeres, as well as quantify the telomeres per cell or per chromosomicgroup. The method called flow FISH uses flow citometry in combinationwith the FISH technique using a pan-telomeric PNA (peptide nucleic acid)as a probe and allows to measure, using the fluorescence intensities,the average telomeric lengths at the chromosome ends in individualcells. For our purpose, the fluoresce intensity of PAN labelled withchromosomes at metaphase. The results are expressed as telomerefluorescence unit (TFU) corresponding each TFU to 1 kb of repetitivetelomeres.

Results

Changes in the average length of telomeres in human fibroblasts culturedunder oxidative stress conditions with or without incorporated DHA wereanalysed by flow-FISH (FIG. 23). A linear regression was used to analysethe relation between the length of telomeres and the pass number ofcellular populations. For all the analysed cultures, the slopes in theregressions can be understood directly as the telomere shortening index.In human fibroblasts, the treatment with AAPH, which induces an excessof intracellular free radicals, accelerate noticeably the telomereshortening index. On the other hand, the incorporation of DHA at aconcentration of 0.5 μM, which has been proved to increase the cellantioxidant defence, reduces said index by 50% in respect to its valuewithout DHA. Furthermore, the incorporation of DHA is capable ofreducing the telomere shortening index, even in respect to the normalcontrol of fibroblasts.

1. (canceled)
 2. A method of treating cellular ageing or inhibitingpremature senescence in human skin, the method comprising administeringdocosahexaenoic acid (DHA) to the human skin, wherein the DHA isincorporated into a glyceride, and wherein a portion of the DHA isincorporated into an sn-2 position of the glyceride.
 3. The method ofclaim 2, wherein the cellular ageing or premature senescence isassociated with cellular damage evidenced by a shortening of DNAtelomeres in cells of the human skin.
 4. The method of claim 2, whereinthe glyceride is a triglyceride and between 40 and 100% by weight offatty acids in the triglyceride is the DHA.
 5. The method of claim 2,wherein the glyceride is a triglyceride and between 66 and 100% byweight of fatty acids in the triglyceride is the DHA.
 6. The method ofclaim 2, wherein the DHA is enzymatically incorporated into theglyceride.
 7. The method of claim 2, wherein the glyceride is formulatedin a cosmetic application.
 8. The method of claim 7, wherein thecosmetic application is in the form of an emulsion.
 9. The method ofclaim 1, wherein the DHA is administered topically to the skin.